The present invention relates to improvements in product yield from fermentation to produce recombinant proteins, particularly in prokaryotic and simple eukaryotic systems. More particularly, this invention greatly increases the yield of properly assembled proteins in large scale fermentations.
The production of large quantities of relatively pure, biologically active polypeptides and proteins is important economically for the manufacture of human and animal pharmaceutical formulations, enzymes, and other speciality chemicals. Recombinant DNA techniques have become the method of choice to produce large quantities of exogenous proteins using bacteria and other host cells. The expression of proteins by recombinant DNA techniques for the production of cells or cell parts that function as biocatalysts is also an important application.
Producing recombinant protein involves transfecting host cells with DNA encoding the protein and growing the cells under conditions favoring expression of the recombinant protein. The prokaryote E. coli is favored as host because it can be made to produce recombinant proteins in high yields. Numerous U.S. patents on general bacterial expression of DNA encoding proteins exist, including U.S. Pat. No. 4,565,785 on a recombinant DNA molecule comprising a bacterial gene for an extracellular or periplasmic carrier protein and a non-bacterial gene; U.S. Pat. No. 4,673,641 on co-production of a foreign polypeptide with an aggregate-forming polypeptide; U.S. Pat. No. 4,738,921 on an expression vector with a trp promoter/operator and trp LE fusion with a polypeptide such as IGF-I; U.S. Pat. No. 4,795,706 on expression control sequences to include with a foreign protein; U.S. Pat. No. 4,710,473 on specific circular DNA plasmids such as those encoding IGF-I; U.S. Pat. No. 5,342,763 on improving expression in bacteria by manipulating oxygen delivery; and U.S. Pat. No. 5,639,635 on secretion of the expressed protein into the bacterial periplasm.
Recombinant proteins become less expensive if the fermentation yield improves. Yield depends upon the rate at which the recombinant protein is properly folded and assembled protein is formed and upon the length of time over which the protein is produced.
The recombinant protein expression rate is typically affected by the growth and metabolic rates of the cells. At higher growth rates, the rate at which a protein can be expressed when induced typically increases (Curless et al., Biotechnol. Prog. 1990, 6:149). However, upon induction, high protein expression rates may not always lead to high rates of formation of active, properly formed products. In other words, while the quantity of protein translated may be maximized, other factors may compromise the quality of the product, such as degradation of the protein by proteases or other detrimental post-translational modifications (Ryan et al., Biotechnol. Prog. 1996, 12:596; Yoon et al., Biotechnol. Prog. 1994, 43:995). Efficient fermentation requires balancing growth rate against yield of usable protein; compromises between these factors result in a decrease of the overall yield below its theoretical potential. Consequently, some intermediate growth rates may be more favorable for the production of high quantities of high quality product.
An added complication is that induction of recombinant protein expression essentially highjacks the cellular protein assembly process to make large quantities of a product with no benefit, and often with significant detriment, to the cell. In fact, for cases in which induction is triggered by phosphate depletion using the alkaline phosphatase promoter, growth rate is dramatically curtailed by the phosphate starvation itself. This effect does not affect the metabolic rate, however.
Thus, there is a need in the art to increase the yield of usable recombinant protein production. The present invention advantageously and unexpectedly addresses this need by permitting high levels of protein synthesis, assembly and folding. Because different factors may play critical roles in maximizing usable protein yield prior to induction during the growth phase of the culture, and post-induction, the independent control of these two factors can lead to improved yields of usable products, such as for the case of soluble, properly folded and assembled antibody fragments.
The invention provides a method for increasing product yield of a polypeptide of interest produced by recombinant host cells, where expression of the polypeptide by the recombinant host cells is regulated by an inducible system. More specifically, the method involves culturing the recombinant host cells under conditions of high metabolic and growth rate, then reducing the metabolic rate of the recombinant host cells at the time of induction of polypeptide expression.
In a specific embodiment the invention provides a method of increasing product yield of an antibody, growth factor, or protease produced by a recombinant E. coli host cell regulated by an inducible system.
In a further specific embodiment, the invention provides a method of increasing the yield of actively folded proteins having different structures, for example Fabxe2x80x22 versus Fab Fv antibody fragments, by selecting the time to initiate reduction in metabolic rate (the rate shift), the rate of adjustment (shift) of the metabolic rate, and the final metabolic rate. Adjusting these parameters of the invention enhances the yield of correctly folded proteins having different secondary and tertiary structures, interaction and refolding characteristics, size and contact area, and other factors that can affect protein assembly and function.